ES2230877T3 - MICRODISECTION OF PRECISION FOR CAPTURE WITH LASER USING SHORT PULSE LENGTH. - Google Patents

Abstract

Laser capture microdissection occurs where the transfer polymer film is placed on a substrate overlying visualized and selected cellular material from a sample for extraction. The transfer polymer film is focally activated (melted) with a pulse brief enough to allow the melted volume to be confined to that polymer directly irradiated. This invention uses brief pulses to reduce the thermal diffusion into surrounding non-irradiated polymer, preventing it from being heated hot enough to melt while providing sufficient heat by direct absorption in the small focal volume directly irradiated by the focused laser beam. This method can be used both in previously disclosed contact LCM, non contact LCM, using either condenser-side (or beam passes through polymer before tissue) or epi-irradiation (or laser passes through tissue before polymer). It can be used in configuration in which laser passes through tissue before polymer with and without an additional rigid substrate. In its preferred configuration it uses the inertial confinement of the surrounding unmelted thermoplastic polymer (and the overlying rigid substrate) to force expansion of the melted polymer into the underlying tissue target. Utilizing the short pulse protocol, the targeted and extracted material can have a diameter equal to or smaller than the exciting beam.

Description

Precision microdissection by laser capture using short pulse length.

Field of the Invention

The present invention relates to the microdissection by laser capture, a technique in which it is displayed a specimen using a microscope and in which it is then cover the specimen with a layer of transfer material that, once activated by a laser, it adheres to the elements of specific destination of the sample by detaching them from it for their further processing More particularly, the present exhibition It focuses on the extraction of samples of equal size or more smaller than the activation laser beam. The objective of the present invention is to provide a method and an apparatus for the reliable microdissection of objectives located within a tissue or other specimen samples, smaller than 10 microns in diameter, approximately.

Background of the invention

In WO 97/13838, entitled "Isolation of Cellular Material Under Microscopic Visualization "and published on April 17, 1997, on page 20, line 24, states the following:

The size of the transferred tissue, depending on the needs of the technician, can be varied by changing the diameter of the laser beam and the duration of the pulse. Highly reproducible transfers in the range of 60 to 700 µm in diameter can be easily made to obtain small lesions (100 µm to 1 mm) without invasion of adjacent non-neoplastic cells. In most basic and clinical research studies, it is necessary to obtain between several hundreds and several thousands of cells to provide enough genetic material and carry out reliable amplification and statistically significant analysis. However, since the laser beams can be focused on an area smaller than a cell diameter, it is considered that transfers of specific individual cells or even parts thereof are possible according to the method of the invention .

In the present application, the solution is exposed for the transfer "considered possible" mentioned more above.

Although in the first microdissection patents an inert rigid substrate to which the polymer is applied is described thermoplastic that can be used as a pressure plate, in the original implementation of the LCM (capture microdissection with laser) an independent film is used that is applied to the tissue surface, gently pushing the film against the sample. Next, the film placed on the section of the desired tissue is heated by a beam of 100 microns in diameter and is melted by CO 2 laser pulses. The length of Laser pulse chosen (between 100 ms and 630 ms) allows the film irradiated increase to a steady state temperature for a long enough time, so that the polymer flow into the tissue and form a strong bond by filling the empty spaces of the dried sample. 630 ms pulses commonly used with this system are chosen with this duration on purpose, to ensure that the molten polymer (which remains molten until the end of the pulse) has enough time, after reaching the steady state temperature, to flow reliably into the tissue during the laser pulse. In a subsequent trial has demonstrated the possibility of carrying out equivalent transfers with this system and 100 ms pulses, although due to the irregular separation between the surface of the polymer and tissue, 100 ms pulse transfer is less reproducible than with longer pulses. When you use this LCM system, the objective is to heat the lower surface of the polymer slightly above the melting point. The levels of energy supplied by the CO2 laser is kept inside by a factor of two of the required threshold energy (range 25 to 50 mW supplied to a 100 µm point of a polymer film EVA 100 µm thick). Therefore, the captured tissue by molten polymer it is usually exposed to temperatures maximum of \90 at 100 ° C for 500500 ms. When it's used In this procedure, no damage to DNA, RNA or proteins of the sample captured in the subsequent analysis molecular.

In LCM, short pulses are avoided to ensure adequate bond strength. The informations provided by a number of adhesive manufacturers EVA-based thermoplastics (e.g. hot glue) suggest that the use of EVA adhesives requires maintaining molten joints under pressure for more than one second. In the Original LC2 laser designs of CO2, using a pressure plate (transparent and non-absorbing laser light and visible light) is not practical, due to the shortage and cost of the materials that transmit laser wavelengths of CO 2 (9 to 11 µm). Subsequently, the introduction of Highly near infrared absorbent dyes (sim0.8 um) soluble in thermoplastic polymers has allowed the transfer film is focally founded by diodes infrared pulse laser (\ sim0.8 \ mum), which are focused with ease through transparent substrates on diameters small ones smaller than 10 µm from the film absorbent thermoplastic.

International application WO98 / 35216, which was published on August 13, 1998 and claims a date of priority of February 7, 1997, is part of the state of the technique only in relation to Article 54 (3) CPE. This document discloses an LCM procedure, in which the transfer film is separated from the sample by a small space interval, but does not describe the observation stage of the optical interface between the sample and the transfer film during the irradiation procedure.

Summary of the invention

The present invention relates to a procedure for direct extraction of material from a sample according to claim 1.

The microdissection by laser capture takes place when the transfer polymer film is placed on a substrate that covers the visualized and selected cellular material to be extracted from a sample. The transfer polymer film is focally activated (melts) with a pulse short enough to allow the molten volume to be confined to the directly irradiated polymer. The present invention uses short pulses to reduce thermal diffusion to the surrounding non-irradiated polymer, thereby preventing it from heating up to melt and providing sufficient heat by direct absorption for the small focal volume directly irradiated by the laser beam. in focus. This procedure can be used both in the LCM described above and in the LCM without contact, either using radiation from the condenser side (the beam passes through the polymer before passing through the tissue) or epirradiation (the laser passes through the tissue before going through the polymer). The procedure can be used in configurations in which the laser passes through the tissue before passing through the polymer, with and without an additional inert substrate. In the preferred configuration, the inert or elastic confinement of the surrounding non-molten thermoplastic polymer (and the superimposed bonded substrate) is used to force the molten polymer to expand into the desired underlying tissue. Using the short pulse protocol, the focused and extracted material can have a diameter equal to or less than that of the exciter beam, even if it is close to the optical diffraction limits.

To further improve accuracy and location, a series of short pulses can be applied "sub-thresholds" to the same points or to points immediately adjacent to come into contact only with specific objectives inside the laser beam (for example, a target lower than diameter of the laser beam located in the center of the laser pulse). In this case, capitalizes on the fact that when a volume of polymer melts from top to bottom in the thermoplastic film absorbed by the action of a laser pulse, it experiences a proportional volumetric expansion in tissue direction. This polymer expansion volume can be matched with the desired target volume, including separation volume initial between the polymer and the target, either by calculation approximate of the average pulse parameters necessary to carry perform the capture with a single pulse, or by means of a laser pulse which is about half of what is necessary for capture with a just impulse and applying a series of pulses until it is achieved the union with the objective.

The objective of the present invention is provide a procedure that allows a transfer zone Reproducible LCM less than 20 microns, with maximum precision and efficiency and minimum duration of thermal transients in the sample destination, caused by contact with the polymer thermoplastic melted during the laser pulse and subsequently Until it cools. It has been proven that a reliable procedure for smaller transfer point sizes (less than 10 microns in diameter) includes the reduction of width of the laser pulse less than 1 ms and the power setting laser maximum These pulse widths and powers reduce the minimum damage to macromolecules of the tissue sample.

It has been verified, at the time of presenting the present request, that the contact and adhesion of the material Activable with the sample creates a perceptible phenomenon. Actually, the user can perceive the desired adhesion with the sample while adjusting the pulse length and the power to dilate, contract and even conform the areas of adhesion.

Brief description of the drawings

Figure 1A is a microdissection scheme for laser capture, in which the activation laser light passes to through the condenser side of a microscope for the microdissection of the selected tissue.

Figure 1B is a detail of the location of the LCM illustrating the laser excitation beam and the ability to excitation of an area less than the beam to apply with greater Precision contact LCM to the selected tissue.

Figure 2A is a contactless LCM scheme, in which epirradiation is used (the laser passes through the tissue before going through the polymer) so that the activated film extend to the selected tissue through a space.

Figure 2B is a detail of the location of the LCM illustrating the laser excitation beam and the ability to excitation of an area less than the beam to apply with greater Precision LCM without contact to the selected tissue.

Figure 3 is a view of an EVA layer (ethylene-vinyl acetate), in which a You pass through the layer.

Figures 4A to 4E are radial profiles successive of the EVA of Figure 3, illustrating how the expansion of the radial profiles exceeds the dimension of the beams with the increased radiation duration, and how it increases the contour interval with increasing beam power of the laser, it being understood that the contours of 25 ° C of a 20 mW laser beam (a 40 mW beam would generate a 50ºC contour in the same way).

Detailed description of the specific embodiments

In relation to Figure 1A, an LCM is illustrated of conventional contact. In Figure 1A, the observer represented by eye E displays the specimen S of the slide P through microscope M (represented schematically). The S specimen lighting is provided through the lamp incandescent of microscope L and dichroic mirror D. La lighting passes through a transparent support B (which can be an inert non-absorbent layer of rigid material or material strong but flexible, such as a polyester film, forming the first type of material a rigid transfer element and the second, in combination with the T layer, a more flexible tape of 2 or more layers) and a transparent transfer layer T.

The transfer layer T is usually a polymer low melting thermoplastic with a large increase in volume associated with the transition from solid phase to liquid phase (e.g., ethylene-vinyl acetate) that can be dyed (e.g., at near infrared wavelengths invisible to the human eye) to attach to laser radiation 16 Z of a specific frequency. When determining which part of the Specimen S should be applied LCM, laser Z is activated and then the transfer layer T is activated to adhere to the specimen S of the selected tissue. Light 16 of the Z laser passes through the lens 14, affects the dichroic mirror D and then goes on to through the transparent support plate B to influence the layer transfer T.

A detail of the LCM is illustrated in Figure 1B of illustrated contact. The laser beam 16 shown passes through the transparent support B and penetrates the transfer layer T (which absorbs the beam but not visible light). The layer of T transfer represented is in contact with the S specimen. In relation to Figures 4A to 4E, it will be understood that it is possible excite an area of the transfer layer T smaller than the total exposed area of it. Therefore, the column of adhesion C represented extends to the inside of the S specimen to extract the group of selected G cells that It can consist of only one cell.

As will be understood, the transparent support B This LCM has a function. In particular, and through support which provides the transfer layer T, the support plate transparent B forms a sufficiently strong underlying substrate as to resist the expansion of molten thermoplastic polymer. This underlying substrate combined with inertial confinement or elastic of the inactivated material surrounding the layer of T transfer forces molten polymer to flow in the direction of the tissue sample. The preferred requirements for the substrate Underlying are: 1) that is transparent to both visible light necessary for microscopic visualization such as laser light infrared used to activate the thermoplastic polymer, 2) that has a melting point high enough for the heat of the adjacent molten laser absorbent thermoplastic polymer no cause its fusion and 3) to be sufficiently rigid so that it does not significantly deform to the pressures caused by the expansion of the adjacent thermoplastic polymer activated by the laser (that is, behave like a rigid body against these forces transitory).

So far, the general description of the type of LCM contact. The type of contactless LCM can be illustrated using Figures 2A and 2B.

Figure 2A is essentially the same as Figure 1A, with the exception that the radiation used is called epirradiation Consequently, as will be seen, the Z laser will place below the slide P.

In relation to Figure 2B, it will be noted that the laser beam 16 ascends, through the slide P, to the shows S and the spatial interval 20. Next, the beam of laser 16 is introduced into the activation layer T to activate it. As will be described, the adhesion column C crosses this spatial interval 20 and again fixed to the selected material G. Also this time, the fixation to the selected material G consists of a column that has a diameter smaller than the actual diameter of the make. Therefore, using the techniques described below, it is possible to "focus" the activated material in relation to the beam of the laser 16 so that it actually occupies an area (or a diameter of transfer destination) lower than the laser beam (diameter), even if the beam diameter is reduced below 10 µm.

In relation to Figure 3, a thermal model of polymer temperature contours (EVA) which makes up the transfer layer T. It will be noted that the layer of superimposed T transfer is the transparent support plate B and that the underlying transfer layer T is the slide of P glass with specimen S. The diameter of the beam is 30 \ mum.

With reference to the darkened area represented in which indicates that the polymer is molten, a important deduction. The reader will understand that before the molten film and specimen come into contact in the part selected, there is an air interface between the specimen and the movie. The air-sample interface is usually a very irregular surface with a large difference in indices of refraction, which causes a large dispersion of the incident light In the sample.

When contact and adhesion of the molten thermoplastic polymer with the sample, the difference of refractive indices are greatly reduced (refractive indices of the polymer and sample are much more similar to each other than indices of any of these and air), and consequently observe a clearly visible change in the transmission image of the sample at the point of contact. Specifically, the dispersion decreases and the contacted and attached part of the specimen looks more "sparkly". Therefore, when union with the molten transfer film, an interface clearly appears Visible to the observer.

It should be noted that this change in appearance is produces even when the movie is already "in contact" with the specimen. In the usual case, the surface of the specimen is very irregular. The contact of the film with the specimen in the points "high" of an irregular surface still generates a light dispersion very noticeable. When the heating and adhesion, the footprint of the joint is relatively easy to observe Therefore, in the procedure disclosed, the technician has the advantage of being able to observe the union at the moment in which it occurs.

Using this technique, the point size can adjust visually using the present exposure. By For example, when accurate dissection is needed, the observer You can visually verify that at no time does the point reach be larger than the selected area. On the other hand, when the desired sample material is essentially isolated (either by a spatial separation or by a "harmless" material that surrounds) the contact area can be enlarged. In any case, the Contact described here provides a valuable visual interface that guides the observer during the collection procedure.

Characterized obscured areas that guide the person who collects the samples during microdissection also They are illustrated in Figure 4.

In the usual implementation of the LCM by near IR absorbent polymer films with an OD of sim0.3 and a thickness of sim50 microns, the pulse duration necessary to approach the steady state in the center of the laser irradiated point is \ sim100 ms for beams of \ sim100 \ mum, \ sim40 ms for 60 \ mum beams, \ sim10 ms for beams 30 µm, \ sim1 ms for 10 µm beams and about 500 us for 5 µm beams.

Using a special LCM system capable of Supply almost high power laser diode pulses of only 200 \ mus, certain characteristics have been quantitatively demonstrated new operating with short pulses, compared to steady state pulses previously used with the LCM, which are the object of the present invention.

For a beam of 10 µm, pulses of length less than 1.0 ms in an EVA response (expansion and joining) which depends on the product of the power and the duration of the pulse (or total energy supplied). In this short pulse regime, the polymer heating efficiency and the creation of a joint is optimal With longer pulses, a larger quantity must be supplied total heat (or absorbed laser energy that is the product of the pulse duration and absorbed power), which determines that the transfer sizes are larger and the molecules of the tissue are exposed to higher temperature integrals by time (that is, longer exposed to high temperatures). In general, for a pulse duration exceeding this regimen of "short time", secure transfers will be longer size and macromolecular thermal transients will be more prolonged Similarly, for 5 µm beams, the pulses lasers of less than 0.5 ms will provide equivalent transfers at a total equivalent energy (power and pulse duration necessary for "effective" transfers vary in a way mutually reciprocal).

The duration of the thermal transient experienced by the macromolecules of the target tissue can be reduced using shorter pulses, obtaining the same maximum temperatures. The size of the transferred tissue is determined by the diameter of the area of the film that is heated to melt and fuse with the tissue sample. For long pulses (that is, under the steady state conditions described by Goldstein et al . In Applied Optics 37: 7378, 1998), the transfer size is determined by the power of the laser beam and by the thermal characteristics (capacity and thermal diffusion) of the LCM transfer film. The table provided below summarizes the parameters for the conventional steady-state LCM, matching the size of the transfer with the diameter of the beam (using a polymer 40 µm thick and a tissue section 5 µm thick):

TABLE 1 Conventional LCM parameters used for 30 to 100 µm transfers

Beam diameter Size of transfer Laser power Pulse duration To be 30 \ mum 30 µm 15 mW 25 ms * 60 µm 60 µm 30 mW 50 ms * 100 µm 100 \ mum 40 mW 100 ms * * greater than or equal to

At higher powers, the size of the transfer and the corresponding maximum temperatures of polymer increase. However, if the widths of pulse above the values indicated in Table 1, nor the diameter of the transferred point nor the maximum temperatures vary from significant shape with pulse width. When necessary fix the LCM transfer film to an inert substrate instead of using an independent film, the substrate:

1) acts as a pressure plate that provides a defined application force,

2) reproducibly delimits the surface lower or binding with the activatable polymer fabric in relation to with a non-activated surface and provides accuracy of rigid body positioning,

3) prevents polymer expansion through the surface of the rigid substrate or along it, and in combination with the inactivated thermoplastic polymeric film (and therefore solid) surrounding forces the molten polymer to expand in the direction of the underlying tissue and enter into this, which results in a surface contact area reproducible between polymer and tissue and a strong bond.

In other words, as long as the laser film with enough absorbed energy to melt it focally, the molten film tries to expand (due to to the coefficient of thermal expansion and the increase in volume associated with the change from solid to liquid), but is confined by the stiffness of the film support and the unheated parts of the movie. Once the "bottom" surface of the polymer activated has melted, the expansion pushes the molten film down by inserting it into the tissue. Therefore it is not it is necessary that the film be in direct contact with the objective. The bottom surface of the molten part of the film is pushed during the laser pulse, fuses or joins with the target microscopic and the target located within the volume delimited by the polymer expansion zone is captured or fixed to the film and substrate. The combination of 1) short beam pulses and small, 2) the great expansion when the fusion of the selected polymers and 3) the confinement of the expansion and redirection to the target allows reliable capture of targets less than 10 µm. For longer pulses (as in the case of Original CO2), the polymer can expand upwards and down and flow by gravity or tension surface once the molten polymer has come into contact with the tissue (or wet surface of the lens). However, for for shorter pulses to form effective junctions, a high local pressure to force the rapid flow of polymer inside of the objective (both if they are initially in contact as if no). The expansion of molten polymer creates this pressure. If he molten volume is confined by the rigid material by all sides, except in the direction of the target, such joints can be done in less than one ms and are very strong. The support (layer of substrate) is particularly crucial when using the irradiation of the "condenser side", in which the part top of the film melts before the bottom. Without inert substrate of "confinement", the initial expansion is will produce in the opposite direction to the target and the pressure that boosts the flow in the direction of the tissue, once it has been produced the merger from top to bottom, it will be considerably reduced Therefore, unions without confinement of the polymer expansion at the top will be less strong or they will form less reliably in short times.

As long as these unions are stronger than the original junction between the lens and the glass slide, the target will be transferred to the surface of the film and separated of adjacent slide elements once the substrate it has been separated from the slide (if there are adjacent elements no desired located next to the desired elements or attached to them, to achieve separation it will be necessary that the bond strength of unwanted items with the glass slide exceed the binding force of the unwanted elements with the elements desired). Usually, a series of laser joints are formed to accumulate a group of equivalent objectives of the desired area in the slides before the separation of the substrate, its polymeric layer thermoplastic and the captured objectives of the slide and the rest of microscopic elements of it.

Preferred embodiment

Any of the samples or films are used Existing LCM with any display geometry or irradiation (eg, condenser side irradiation or epirradiation). The combination of 1) short beam pulses and small, 2) the large fractional expansion at the time of the merger of the selected polymers (e.g., 1010%) and 3) the confinement of expansion and redirection towards the objective allows reliable capture of targets below 10 µm. For longer pulses (as in the case of original CO2), the polymer can expand up and down and flow through gravity or surface tension action once the molten polymer has come into contact with the tissue (target porous). Shorter laser pulses at somewhat more powers are required higher than those of conventional LCM to confine volume molten polymer fixed to small beam diameters (<20 microns) For these shorter pulses, a local pressure is required high to force the rapid flow of polymer into the target (whether they are initially in contact or not). The great Molten polymer expansion creates this pressure. If the volume cast is confined by rigid material on all sides, except in the direction of the objective, said joints can be formed in less than one ms and they are very strong. The support (substrate layer) is particularly crucial when using "side irradiation" of the condenser. "In this case, the top of the film it will melt before the bottom, and the initial expansion without an inert "confinement" substrate will occur in the opposite direction of the target. This fluid path to the air at the top will significantly reduce the pressure that boosts the flow in the direction of the tissue once it has had Place the fusion from top to bottom. Therefore, the unions will not be so strong nor will they form so reliably in times short

Unlike the long pulse LCM conventional, at pulse widths below 1 ms, the film does not approaches thermal equilibrium at distances of more than 2 microns from edge of the laser beam during the laser pulse. Consequently, the Heat loss is minimized. Therefore, when the layer activatable receives and absorbs adequate energy, only the film of the area exposed to the laser is heated to the melting point during the laser pulse, expanding to come into contact and merge with the tissue. For a short pulse (less than 1 ms), no sufficient time is available for the temperature of the film increases to the melting point outside the exposed area to the laser pulse, due to the short duration of the pulse. This zone remains below the melting point and does not merge with the tissue. Therefore, for short pulses, the size of the area of transfer is not increased by thermal diffusion and the size of tissue transfer is determined primarily by the diameter of the laser point. For short pulses, it is observed that the transfer point size increases when the width of the laser pulse, in contrast to the LCM pulse regime conventional lengths, which indicates that you are working on a different thermal diffusion regime. The table provided to Below summarizes the parameters for the new short pulse LCM with a transfer size equal to the beam diameter (using a polymer 40 µm thick and a tissue section 5 µm thick).

TABLE 2 Short pulse LCM parameters needed to transfers of smaller sizes (5 and 10 µm diameter of 5 µm thick targets

one

It is important to note that the union obtained from EVA polymers (such as Dupont Elvax 200W, 410, 205W and 4310) with the fabric it is reliable and has sufficient strength to allow reproducible transfers in thermal transients of only 0.3 ms. Therefore, it is not only possible to confine the polymer fused to an area of a few microns in diameter with short pulses, but also form reliable bonds with the tissue in said short melting transients.

In table 2 above, the measurements experimental demonstrate equivalent transfers for it pulse energy when the duration of the laser pulse is maintained by below a critical time associated with the time needed for a significant thermal diffusion from the irradiated volume. How I know has indicated, below a certain pulse length (<< 1 ms for a point of 10 µm in diameter and 0.5 ms for a point of 5 diameter), the laser energy needed to create a size Small transfer given is essentially constant (eg, 9 microjoules for a 5 micron transfer using a EVA film 40 µm thick with an OD of 0.4 at laser wavelength). In general, in the realizations Preferred LCM thermoplastic polymers are used (e.g., from EVA with low percentage of vinyl acetate) with a temperature of low melting, so that the maximum temperatures required to form a thermoplastic joint are minimized.

As can be seen in Table 2, in the Short pulse rate used, the laser energy needed to creating a small transfer size given is essentially constant (e.g., 9 microjoules for a 5 micron transfer using an activatable EVA film 40 µm thick with an OD of 0.4 at the wavelength of the laser). Although it is possible use any of these pulses to capture an element microscopic of equivalent size, there are certain differences intrinsic physicists who advise using strategies of Concrete optimization In general, the shortest pulses of higher power (eg, 5 µm, 45 mW and 0.2 ms with 9 microjoules) create maximum temperatures in the thermoplastic polymer higher than those of the equivalent longer pulses (eg, 5 um, 18 mW and 0.5 ms also with 9 microjoules). When the temperatures are increased within the molten polymer, a lower transient viscosity and one more injection may occur effective polymer within the finest empty spaces of the target volume However, the shortest extra pulses power are also associated with maximum thermal transients plus high in the captured objectives, during the brief period between instant when the molten polymer first enters contact with the target and the moment when the polymer cools and solidifies (after finishing the pulse and rapid cooling by heat flow through the underlying glass). On the other hand, the slightly longer pulses of lower power provide the same maximum volume of molten polymer and expansion volume with the same energy absorbed, by lower maximum temperatures and longer times that can minimize thermal attack maximum to the sample, while providing longer times for create a stronger mechanical bond with the objective. In general, in preferred embodiments of LCM polymers are used thermoplastics (eg, EVA with low percentage of acetate vinyl) of low melting temperature, so that the maximum temperature needed to form a thermoplastic junction is minimizes When working with sensitive materials thermally, such as proteins in which it is desired to preserve Enzymatic function, "equivalent" laser parameters Preferred in Table 2 are the most effective effective pulses cited.

The requirements to obtain a satisfactory union of the objective are: 1) the fusion of the activatable polymer along the axis of the laser beam from the upper to the lower surface and 2) the expansion of the thermoplastic polymer in this axis to the target surface and within the empty spaces of destination. In the above LCM, longer pulses capable of providing steady state temperature distributions within the molten polymer, and a thickness of the polymer approximately equal to the laser beam diameter are used (Table 1: from 30 to 100 µm). When short pulses are used to minimize lateral thermal diffusion and allow transfers of smaller targets, the heat flow from the top to the bottom of the film is also reduced during the laser pulse. The thermal gradient induced by the laser from the top to the bottom of the irradiated volume increases both with the thickness of the activatable polymer and with its optical density (absorbance) at the laser activation wavelength. Therefore, in particular for the short pulse LCM, the activatable polymer film must be carefully designed in terms of thickness and optical density. As described above (Bonner et al ., Science 278: 1431, 1997 and patents ...), the absorbance of thermoplastic polymers, such as ethylene-vinyl acetate, at near IR laser wavelengths can be controlled precisely by concentrating added molecules with high near IR absorbance, such as naphthalocyanine dyes that are very soluble in the polymer. The absorbance of the thermoplastic film at the laser wavelengths used must be maintained at an OD <0.43 by varying the concentration of dye with the thickness of the film to be used. Other thinner films are associated, when melted from top to bottom in small dots, with respectively small volumes of molten polymer and, consequently, smaller polymer expansion volumes. In this way, thinner polymeric films will intrinsically have a higher capture accuracy, in particular, for thinner target specimens. On the other hand, when the initial separation space between the non-activated polymer surface and the target surface or the thickness of the target increases, the expansion distance necessary to create a joint and an effective associated transfer also increases. In such cases, the thickness of the polymer should be increased and the concentration of the dye should be reduced.

In another preferred embodiment, the Short pulse LCM procedure at slightly lower powers (of a factor of two of the power required to transfer a object of the specimen exactly equal to the size of the beam with a single pulse), but with a series of pulses (with a repetition of << 2 to 3 pulses per second). This series of pulses increases the advance of the polymer expansion until it comes into contact and joins a cell or target microscopic object located in the slide, at which time the pulse train is interrupted. In this procedure, the first short pulse determines that the polymer fuses focally and separates from the surface by combination of thermal expansion and inert confinement and elastic of surrounding solid materials. Since the pulse individual is short and the cooling / solidification of the extension small is very fast (<< 1 ms), after a brief cooling, it forms a small solid pedestal that protrudes from the surface polymeric Each additional pulse will expand the polymer in the direction to the target in smaller and smaller increments. In this way, for carry out the smallest possible transfers so reliable, just supply the number of pulses needed to expand the polymer until it comes into contact with the tissue. Additional pulses supplied after contact allow the expansion of the polymer inside the tissue until it reaches the size of the laser beam.

Since under microscopic observation during The LCM is observed optical brightening in the target area due to the correspondence of indices between the surface of the tissue and contact polymer, it is easy to determine the first pulse that comes into contact with the tissue. In this way, the transfer can be done safely on a scale that is smaller than the beam size (e.g., it is possible to focus and transfer objects of? 1 micron with a beam of 5 microns).

In a third preferred LCM procedure of short pulse, various additional supraumbral pulses are used after the first contact with the target sample to allow capture of a single irregularly complete cell (subject to sample destination) using a beam that is slightly less than the individual cell. Since the polymer flows into the tissue desiccated porous, with short pulses close to the threshold the polymer flows preferably along macromolecular structures contiguous of a given cell and tends to fill the cell by complete, before expanding through the intercellular edges. In this way, short pulses close to the threshold are capable of focus on individual cells or densely structured connected, such as the cell nucleus, even if they have an irregular shape and do not match the beam shape of the To be.

The accuracy of the short pulse LCM or size minimum of the captured element described here refer to dense specimens, such as a tissue section in which Desired objectives are immediately contiguous to unwanted ones. It should be understood that accuracy is still possible greater when smaller particles are captured with a separation greater than that of its diameters (eg, a dilute cytological smear or a chromosomal crush). In this case, they can be captured smaller pure target elements by expansion polymeric covering the elements and the surrounding empty space of the microscope slide, without union with element no desired closer. Therefore, it is possible to focus and capture microscopically elements of only 1 µm, with great purity (eg, using the 5 um laser pulses in Table 2). Though the final resolution of the LCM capture can be considered as limited by the wavelength of the light used for the visualization and microscopic approach and by the laser of activation, under certain circumstances, by this technique is possible to focus and capture (i.e. purify from a complex mixture) submicron particles. For example, bookmarks specific fluorescence can identify particles specific micronics without solving their structure. Naps particles spread to a sufficiently low density over the microscope slide (approximate mean separation of 5 um), the short pulse LCM can focus and capture them. Further, the images of the rest of the specimen slide and the transfer surface obtained after transfer can Verify and quantify the capture procedure.

The previous LCM technique has been described with Regarding biological applications. It should be understood that These techniques are applicable to any sample that may be observed under a microscope, such as a heterogeneous group of elements in which the procedure causes the separation of a component or group of microscopically recognizable components. Thus, a separation procedure based on the use of a microscope in which a film of transfer selectively activated and applied to a tissue, cytological specimens, cell organelles, chromosomes or viruses. In addition, it is also possible to separate identifiable inert objects by a microscope in subgroups that will be isolated by LCM procedures.

US Patent Application Serial No. 08 / 883,821 titled "Convex Geometry Adhesive Film System for Laser Capture Microdissection ", now US Patent No. 6 100 051, was published on August 8, 2000 and not part of the state of the art. In this exhibition describes the use of a surface convex that has a selectively activatable adhesive, for side-by-side grouping and specimen concentration joined by laser microdissection. How will you observe the reader, in the present exhibition the union of extremely small or scarce elements by microdissection by laser capture Thus, the combination of these two exhibitions will allow isolation and union of elements very small concretes in a group of a microscopic specimen. TO then the transfer of the groups can be carried out formed to any precise place for further analysis.

Claims (8)

1. Procedure for the direct extraction of material of a sample, comprising the following stages: provision of a sample; provision of a transfer film that, only after being activated in selected areas, does it present the property to provide the selected areas with the ability to adhere to the sample; juxtaposition of the sample to the film of transfer; identification of at least part of the material to extract it from the sample; focus of the pulsed radiation beam, of a preselected beam diameter and pulse length preselected, on the transfer film to activate a volume of the transfer film adjacent to the sample and adhere it to at least part of the material to be extracted of the sample; observation of the optical interface between the Sample and transfer film during the focus stage to verify that said part of at least the material of the Sample adhere to the transfer film, and the scope spatial of the union in the image plane, and separation of the transfer film from the shows while maintaining adhesion between the film transfer and said part of at least sample material, to thereby detach said part of at least material from the sample of the remaining part of the sample; in which the transfer film separates a small spatial interval of the sample and in which the beam of pulsed radiation, of a pre-selected beam diameter and a preselected pulse length, focuses on the film of transfer to activate a movie volume of transfer adjacent to the sample, which determines that said volume expand and cover the small spatial interval to adhere to said part at least of the material to be extract from the sample. 2. Method according to claim 1, in the that the transfer film is a thermoplastic polymer with a large volumetric expansion associated with fusion, destined to create an expansion and a sufficient driving force to push the polymer against the target element and insert it into the same and thereby obtain a strong bond quickly. 3. Method according to claim 1 or the claim 2, further comprising the step of: provision of an inert support substrate on the upper face of thermoplastic polymer that prevents the large volumetric expansion associated with fusion deviates from the target desired, and determines that the internal pressure created by the polymer that expands push the molten polymer against the element of destination and enter it to provide a union Strong with the goal. 4. Procedure according to any of the claims 1 to 3, further comprising the step of: increase in pulse length preselected to increase the activated area of the film transfer or reduction of preselected pulse length to reduce the activated area of the transfer film and, optionally, supply of additional pulses after the first pulse, which creates a union to positively increase the diameter of the area attached to a desired target. 5. Procedure according to any of the claims 1 to 4, further comprising the steps following: pulse duration setting to activate a volume of the transfer film with the beam diameter preselected and adhere to that part at least material of the sample, and observation of the optical interface between the Sample and transfer film during the focus stage to verify that said part of at least the material of the Sample adhere to the transfer film, and the scope space of the union in the plane of the image. 6. Method according to claim 5, which It also includes the stage of: pulse duration setting to activate a area of the transfer film less than or equal to the diameter of you have preselected and adhere it to that part for at least Sample material. 7. Method according to claim 5, which It also includes the following stages: decrease in pulse energy, for example, reducing the pulse length or pulse power, such so that a single subthreshold pulse is insufficient to activate the transfer film at any point in the sample, and application of a series of said impulses sub-thresholds to the transfer film that is juxtaposed to said part of at least material to be extracted, without entering in contact with it, until the transfer film is bind to said part at least material to be extracted. 8. Method according to claim 1, which It also includes the following stages: observation of the contact between the film of transfer and said part of at least material from the sample; focus of another beam of radiation pulsed on the sample contact to cause only adhesion between the transfer film and sample, and separation of the transfer film from the shows while maintaining adhesion between the film transfer and said part of at least sample material, such that said part of at least sample material detach from the remaining part of the sample, including said procedure optionally the focus stage of another beam of pulsed radiation on the sample contact to expand the Adhesion area between the transfer film and the sample.